Uncoupling protein 2 regulates myocardial apoptosis via the diabetogenic action of streptozotocin

doi:10.4236/jbise.2011.47064

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J. Biomedical Science and Engineering, 2011, 4, 506-510

doi:10.4236/jbise.2011.47064 Published Online July 2011 (http://www.SciRP.org/journal/jbise/

JBiSE

Published Online July 2011 in SciRes. http://www.scirp.org/journal/JBiSE

Uncoupling protein 2 regulates myocardial apoptosis via the

diabetogenic action of streptozotocin

Xiu-Zhen Li1, Ruo-Yun Tan2, Xiang Lu1＊

1The First Clinical College, NJMU, Nanjing, China;

2The Second Clinical College, NJMU, Nanjing, China.

Email: [email protected], [email protected]

Received 10 January 2010; revised 26 April 2010; accepted 24 May 2011.

ABSTRACT

Objective: Determine the role of uncoupling protein 2

(UCP2) in the myocardial apoptosis of diabetic melli-

tus（DM）. Methods: DM animal models were induced

by streptozotocinon (STZ) on UCP2 knock-out mice

(UCP2KO) and wild-type mice (WT), which were

reared for 7 and 28 days after successful modeling,

respectively. The expressions of relative protein for

myocardial apoptosis, pro-caspase-9, were investigated

using western blot. However, the terminal deoxynu-

cleotidyltransferase-mediated dUTP-biotin nick end

labeling (TUNEL) was used to explain apoptosis at the

DNA level. Results: Image analysis showed that the

expression of pro-caspase-9 protein levels increased

slightly in UCP-/- + DM-7-day group comparing with

DM-7-day group (P > 0.05). The expression of

pro-caspase-9 protein levels increased significantly (P

< 0.05）in UC P-/- + DM-28- day group comparin g with

DM-28-day group. TUNEL analysis indicated that

UCP2 reduced the number of apoptotic myocytes in

the DM-28-day group by 70% in comparison to

DM-7-day group by 30% (P < 0.05). Conclusion UCP2

may be one of the most important factors that con-

tribute to the myocardial apoptosis of DM.

Keywords: Uncoupling Protein 2; Diabetes;

Myocardium; Apoptosis

1. INTRODUCTION

An inner mitochondrial protein known as uncoupling pro-

tein 2 (UCP2) is a member of the uncoupling protein fam-

ily and belongs to the inner mitochondrial membrane an-

ion-carrier superfamily. UCP2 can be detected in the heart

[1], brain, lung, spleen, kidney, liver, and adipose tissue.

UCP2 is a regulator of reactive oxygen species during

electron transport in the mitochondrial inner membrane,

plays an active role in the prevention of atherosclerosis [2],

is one of the etiologies of type 2 diabetes [3], participates

in inﬂammation [4], and regulates cell apoptosis [5]and

aging [6]. The purpose of this study was to investigate the

relationship between UCP2 and the myocardial apoptosis

of diabetic mellitus, demonstrate the important role of

UCP2 in diabetic cardiomyopathy (DC), and provide ad-

ditional theoretical foundation for DC therapy.

2. MATERIALS AND METHODS

2.1 Animals

Male, 8- to 12-week old WT and UCP2KO littermate

mice were used in this study. UCP2KO and WT mice

were generated on a C57B/6 mixed background. The

WT mice were purchased from the Shanghai Silaike

Laboratory Animal Company. UCP2KO mice were pre-

viously generated by homologous recombination in em-

bryonic stem cells on a C57BL/6 mixed background in

the Laboratory Animal Center of Nanjing University,

wherein their genomic loci were analyzed using PCR, as

previously described [7]. The WT and UCP2KO mice

were raised in a colony at the Nanjing Medical Univer-

sity, wherein all of the animal protocols were approved

by the Animal Care Committee, and the animals were

handled according to the guidelines of the Chinese

Council of Animal Care.

2.2. Chemicals and Reagents

STZ (N-(methylnitrosocarbamoyl)-α-D-glucosamine),

anti-caspase9 antibody, and a TUNEL Apoptosis Assay

Kit was obtained from Bioworld.

2.3. STZ-Induced Diabetes

Each mouse received a single daily injection of STZ (60

mg/kg) for three consecutive days. Animals in the nega-

tive control group (NC) and blank control group (BC)

were identically handled, but were injected with only

vehicle (citrate buffer). All STZ-Injected animals were

assessed for the development of diabetes as described

previously [8] and sacrificed on days 7 or 28, and their

tissues were harvested for further analyses.

X.-Zhen Li et al. / J. Biomedical Science and Engineering 4 (2011) 506-510 507

2.4. Blood Glucose Concentration

Measurements

Plasma glucagon concentrations were assayed using a

glucometer (Johnson & Johnson) according to the

manufacturer’s instructions.

2.5. Western Blotting

Heart tissues were immediately homogenized in Lysis

Buffer at 4˚C as described previously [9]. Insoluble ma-

terial was removed by centrifugation for 30 min at

14,000 rpm at 4˚C. The protein concentration of apop-

tosis protein was measured using a bicinchoninic acid

(BCA) kit and standardized for 60 μg/ml. The total pro-

tein was separated by 12% separating gel with a 3.9%

stacking gel. Next, the gel was electro transferred onto

nitrocellulose membranes (Trans-Blot, Bio-Rad) and

blocked with 5% skim milk for 2 h. The membrane was

incubated in primary antibody (anti-pro-caspase-9, 1:600)

and diluted in 0.1% Tween Tris-buffered saline (TBST)

overnight at 4˚C. Subsequently, the membrane was in-

cubated in secondary antibody (anti-rabbit IgG HRP

conjugate, 1:800) and diluted in 0.1% TBST for 1 h at

room temperature. Specific signals were detected using

enhanced chemiluminescent (ECL) plus reagent (Bio-

Rad). The protein loading was 30 μl and was normalized

using a housekeeping gene antibody (mouse anti β-actin

at 1:10000 and anti-mouse IgG HRP at 1:5000).

2.6. Apoptosis and Immunohistochemistry

Small (<1.0 cm) pieces of heart remnants were ﬁxed

overnight at 4˚C in a solution of 4% paraformaldehyde

in phosphate-buffered saline. The tissue was dehydrated,

paraffin-embedded, sliced (4-μm thickness), dewaxed,

and hydrated. Apoptosis was assessed via the TUNEL

method using a ApopTag ﬂuorescein in situ detection kit

(Bioworld). Labeling indices were determined without

previous knowledge of the genotype of the mice by

counting the number of TUNEL-positive cells per 40

myocytes in each field. There were at least five fields for

each slice and six slices for each animal.

3. STATISTICAL ANALYSIS

The data are expressed as mean ± S.E.M, where applica-

ble. The significance of the data was evaluated using

univariate analysis via the Tukey-Kramer test or Dunn’s

multiple comparisons post test, where applicable. Dif-

ferences were considered as being statistically signifi-

cant at P < 0.05.

4. RESULTS

4.1. The Effect of UCP2 on the Development of

Hyperglycemia in STZ-Treated Mice

WT and UCP2KO were compared to evaluate how the

absence of UCP2 affects the development of hypergly-

cemia. Blood glucose was measured at the time of suc-

cessful modeling, in order to monitor the development of

the STZ-induced hyperglycemia. Both control (normal

saline only) groups exhibited consistent blood glucose

homeostasis over the 7-day and 28-day study periods

and did not show any significant difference or change in

blood glucose concentration in comparison to their

starting values. After successful modeling, the blood

glucose levels in both the WT and UCP2KO mice were

not significantly different; however, after day 28, STZ-

treated UCP2KO mice demonstrated less hyperglycemia

development in comparison to WT mice (Figure 1).

4.2. The Effect of UCP2 on Myocardial

Apoptosis after STZ Treatment

Myocardial apoptosis was measured via Western blot

analysis of apoptotic protein and TUNEL staining on

paraffin sections.

The expression of pro-caspase-9 was observed to in-

crease in the UCP-/- + DM-7-day group in comparison

to the DM-7-day group, although this increase was not

statistically significant (P > 0.05), whereas the expres-

sion of pro-caspase-9 was observed to increase in the

UCP2-/- + DM-28-day group in comparison to the

DM-28-day group, wherein these results were statisti-

cally significant (P < 0.05, Figure 2). The number of

TdT-labeled myocytes decreased 2.0-fold, from 71.00 ±

0.087% in the UCP2KO + DM-28-day group to 30.10 ±

0.079% in the DM-28-day group (P < 0.05), whereas

decreased 1.2-fold, from 12.98 ± 0.050% in the

UCP2KO + DM-7-day group to 9.84 ± 0.079% in the

DM-7-day group (P > 0.05) (Figure 3).

5. DISCUSSION

DM is a major risk factor for the development of several

cardiovascular complications, which in turn have be-

Figure 1. Blood glucose levels in the

different groups, for A, B, C, D, E, F,

X.-Zhen Li et al. / J. Biomedical Science and Engineering 4 (2011) 506-510

508

Figure 2. The expression of pro-caspase9 at different time (7-day, 28-day) in different

groups, for A, B, C, D, E, F, G.

(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 3. The result of TUNEL (10 × 40), respectively, A: NC7-day；B: DM7-day; C：U-/-7-day; D:

UCP2-/- + DM7-day E:NC28-day; F: DM28-day; G: U-/-28-day; H: UCP2-/- + DM28-day; *P < 0.05

versus wild-type.

come the primary causes of death in the diabetic popula-

tion [10]. Hyperglycemia is considered to be the primary

cause of most chronic diabetic complications, such as

cardiovascular pathological changes, diabetic retinopa-

thy, diabetic nephropathy, diabetic foot, etc., which oc-

curs via the deregulation of β-cell insulin secretion and

the development of peripheral tissue insulin resistance.

DC was ﬁrst reported in 1972 by Rubler et al. [11],

who reported the autopsy data obtained from four pa-

tients with diabetic renal microangiopathy and dilated

left ventricles in the absence of other common causes.

Therein, they observed that there was a close relation-

ship between myocardial apoptosis and the occurrence

or development of diabetic cardiomyopathy. In the mi-

tochondrial-initiated pathway, mitochondrial cytochrome

c release and the activation of caspase play important

roles in diabetes-induced cardiac cell death. Caspase

activation is triggered by the formation of a multimeric

Apaf-1/cytochrome c complex that is fully functional in

the recruitment and activation of pro-caspase-9, resulting

in further apoptosis.

In order to further investigate the role of UCP2 in the

development of diabetes, we treated both WT and

UCP2KO mice with STZ injections as to experimentally

induce diabetes. We observed that the UCP2KO mice

developed hyperglycemia, although significantly less

severe in comparison to the WT group.

After four weeks, the myocardial mitochondria of DM

mouse began to swell and denature [12]; On days 7, 14,

and 21 after a single dose of STZ (150 mg/kg) treatment,

the number of TUNEL-positive cells and the amount of

caspase-3 activation increased [13]; Twenty-eight days

after successful modeling (STZ treatment, 60 mg/kg),

cardiac apoptosis in the Sprague Dawley（SD) rats were

observed to have increased (P < 0.01) [14] ; The number

of cardiac myocytes,which cultured by different blood

glucose level, associated with blood glucose concentra-

tion; and Hyperglycemia through mitochondrial pathway

resulted in myocardial apoptosis [15]. Our previous

work has indicated that, even though the expression of

X.-Zhen Li et al. / J. Biomedical Science and Engineering 4 (2011) 506-510 509

cleaved-caspase-3 increased, in addition to cell shrink-

age and nuclear condensation in the DM-7-day group in

comparison to the WT group , this increase was not sta-

tistically significant (P > 0.05), which is consistent with

our reported experimental data.

UCP2 plays a significant role in the onset of diabetes,

specifically via the regulation of cellular ATP produc-

tion [16], islet beta-cell apoptosis [17] and reduction

ROS production [18]. UCP2 also affects the occurrence

or development of DM and participates in the myocar-

dial apoptosis of DC. UCP2KO developed lesser hyper-

glycemia than their WT counterparts after multiple

low-dose STZ injections in vivo [19]. TUNEL analysis

indicated that the number of apoptotic myocytes in the

UCP2KO + DM28-day group decreased by 30% in com-

parison to in the DM-28-day (P <0.05), while the num-

ber of apoptotic myocytes in the UCP2KO + DM7-day

group decreased by 0.2% in comparison to the

DM-7-day (P > 0.05). Our previous work has indicated

that cleaved-caspase-3 expression markedly decreases in

UCP2KO + DM-28-day mice，wherein chromatin was

condensed with high intensity fluorescence in compari-

son to DM-28-day mice, with a statistical significance (P

< 0.05); however, the expression of the cleaved-cas-

pase-3 slightly decreased in the UCP2-/- + DM-7-day

group, wherein chromatin was condensed with low in-

tensity fluorescence, in comparison to the DM-7-day

group, although the results were not statistically signifi-

cant (P > 0.05). Therefore, it can be concluded that

UCP2 reduces the number of apoptotic myocytes and

can provide time-dependent anti-apoptosis activity via

variable caspase-3 or caspase-9 expression.

In this article, myocardial apoptosis slightly increased

in the negative control group in comparison to the blank

group, although this increase was not statistically sig-

nificant (P > 0.05). Although cardiac mitochondrial

UCP2-mediated uncoupling respiration was impaired,

short-term mitochondrial efficacy was increased, which

(7-day or 28-day) improved the function of the myocar-

dium.

Diabetic cardiomyopathy results in myocardial dys-

function. Meanwhile，it has been reported that the loss of

myocardial cell quality and quantity could act as an im-

portant factor in heart dysfunction because they are

non-renewable. UCP2 plays a crucial role in mitochon-

drial energy metabolism and in the occurrence or devel-

opment of DC. Furthermore, it is reported that UCP2 is

involved in determining lifespan [20]. Further research

into UCP2 should investigate the relationship between

UCP2 and myocardial apoptosis, and therein, attempt to

identify the causes so as to improve treatment protocols

and provide a more developed basis for the diagnosis

and treatment of DC.

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